Simplified surgical technique for harvesting corneal stem cells

A specialist details a manual harvesting method that uses instruments available to most anterior segment surgeons.

Thomas John

Ocular stem cell deficiency requires surgical management to restore the compromised cornea. Negation of the stem cell barrier effect — due to systemic or ocular disease, contact lens wear, trauma, or an idiopathic or iatrogenic cause — provides an open pathway for conjunctival invasion of the human cornea. Conjunctivalization of the cornea results in corneal vascularization and chronic keratitis with optical degradation of the cornea and can lead to several unwanted ocular symptoms.

Non-ophthalmic adult stem cells can be harvested from areas including bone marrow and peripheral blood. An adult stem cell is an undifferentiated cell found among differentiated cells, within a tissue or organ, that has the potential to renew itself and differentiate, to yield the major specialized cell types of the tissue or organ.

In this column, Dr. Bouchard describes an economical way of harvesting ocular limbal stem cells using instrumentation that is often readily available.

– Thomas John, MD
OSN Surgical Maneuvers Editor

Normal human corneal stem cells, which are located in the basal layer of the limbal epithelium, are thought to provide a renewable source of corneal epithelial cells. These cells can be irreversibly damaged following thermal or chemical injury, or from hereditary reduction of these cells as in aniridia. Compromise of these stem cells can cause the conjunctival epithelium to migrate over the corneal surface along with goblet cells and neovascular elements, resulting in an opaque, irregular and often painful corneal surface.

Although mild focal stem cell deficiency may be managed by sequential corneal epithelial debridement — allowing the normal epithelial cells to repopulate the abnormal corneal surface — more severe stem cell loss requires surgical replacement of the stem cells. Severe monocular stem cell loss may be managed by transplantation of autologous limbal cells from the healthy fellow eye or homologous limbal cells from a living related donor. In non-autologous allograft transplantation, systemic immunosuppression is required for graft survival.

Many surgical techniques have been proposed to harvest corneal stem cell grafts. These include a variety of manual dissection techniques, the use of mechanical devices and, more recently, the use of femtosecond laser technology. Donor grafts may also be secured using fibrin glue to further facilitate the surgery.

We present a simple, manual, surgical technique to harvest stem cells using inexpensive instrumentation available to most anterior segment surgeons.

Surgical technique

This technique requires an irrigation bulb, a No. 66 Beaver crescent blade, 22-gauge needles, a corneal trephine blade and 0.12 mosquito forceps.

The donor cornea is removed from the Optisol-GS (Bausch & Lomb) storage media and placed over a small amount of gauze for support. The donor cornea is then secured to a water-filled irrigation bulb with six to eight 22-gauge needles (Figure 1). Excess conjunctiva from the perilimbal area is excised. Next, a partial-thickness central corneal trephination is performed using either a Hessburg-Barron suction trephine or a Weck trephine blade that is held manually. The diameter of the disposable trephine is 8 mm to 8.5 mm. A partial-thickness lamellar dissection is then performed using 0.12 fixation forceps and a No. 66 Beaver blade (Figures 2 and 3). To reduce the likelihood of too thin a dissection and inadvertant incision through the limbal tissue, multiple central to peripheral incisions are made using a sweeping motion of the blade. This multipass dissection results in less stretching of the tissue circumferentially, unlike a progressive circumferential dissection technique (Figure 3).

Figure 1. A 4" × 4" gauze is placed under the donor cornea for support, and the donor cornea is pinned to a water-filled irrigation bulb with six to eight 22-gauge needles.

Figure 2. Partial-thickness lamellar dissection is carried out, making multiple central to peripheral incisions. Images: Bouchard C

Figure 3. Higher magnification of lamellar dissection of the limbal tissue.

Figure 4. After 360° of lamellar dissection, the stem cell ring is then excised with Vannas scissors.

Figure 5. Excised limbal lamellar graft.

Following 360° of dissection, the limbal graft is excised using Vannas scissors (Figure 4). The 360° of limbal tissue with only a single break is placed in balanced salt solution (Figure 5). The entire length of the excised ring of limbal tissue is then used in conjunction with half of the second tissue, leaving only two gap areas rather than the three breaks associated with the more traditional grafting of three semicircular pieces of limbal tissues. This may reduce the migration of conjunctive through the "gap" areas.

References:

• Dua HS. The conjunctiva in corneal epithelial wound healing. Br J Ophthalmol. 1998;82(12):1407-1411.
• Holland EJ. Epithelial transplantation for the management of severe ocular surface disease. Trans Am Ophthalmol Soc. 1996;94:677-743.
• Lee D, Kim JH, Choi SK. Femtosecond laser creates thin corneal graft for limbal stem cell transplantation. Ocular Surgery News. 2009:27(2):48-51.
• Meisler DM, Perez VL, Proudfit J. A device to facilitate limbal stem cell procurement from eye bank donor tissue for keratolimbal allograft procedures. Am J Ophthalmol. 2005;139(1):212-214.
• Pandya HK, Djalilian A. The outcomes of limbal stem cell transplantation using fibrin glue. Federated Symposium on Eye Banking and Cornea; October 2009; San Francisco, CA.

• Thomas John, MD, is a clinical associate professor at Loyola University Chicago and is in private practice in Tinley Park and Oak Lawn, Ill. He can be reached at 708-429-2223; fax: 708-429-2226; e-mail: tjcornea@gmail.com.
• Charles Bouchard, MD, can be reached at 708-216-8643 or cboucha@lumc.edu .